| The hallmarks of cancer comprise ten biological capabilities acquired during the multistep development of human tumors.They include sustaining proliferative signaling,evading growth suppressors,avoiding immune destruction,resisting cell death,enabling replicative immortality,promoting tumor inflammation,inducing angiogenesis,genome instability and mutation,deregulating cellular energetics and activating invasion and metastasis.With the development of modern medical technology,the treatment methods of tumor such as surgery,radiation therapy,chemotherapy and biological targeted therapy are continuously improving.Based on latest global cancer statistics(CA:a cancer journal for clinicians,2015),there were still about 14.1 million new cancer cases and 8.2 million deaths in 2012 worldwide.Over the past few decades,nanomedicine has emerged as a promising approach for deliver anticancer therapeutics to tumors due to its preferential and selective accumulation at tumor sites via the enhanced permeability and retention(EPR)effect.Up to now,a dozen nanomedicines have been approved for early stage clinical trials.However,owing to the heterogeneity of the tumor and biological barriers that hinder the effective penetration of nanoparticles,the efficacy of nanomedicines in clinical practice is not satisfactory.The size of nanoparticles is a key factor in the accumulation and penetration of tumor sites.Larger nanoparticles with long-term blood circulation are beneficial for accumulation at the tumor site through EPR effect,while the smaller nanoparticles have a stronger diffusion capacity,which is beneficial for penetrating into the tumor to obtain a better uniform tumor distribution.Therefore,for high enough accumulation and deep enough tumor penetration,an ideal nanocarrier should be size switchable.Here,we developed a temperature sensitive multifunctional nanosystem(TSNCs)loaded with a near-infrared(NIR)agent and a chemotherapeutic drug by an automated assembly method.The TSNCs was about 40 nm,and could be efficiently accumulated on the tumor site.The optical properties of the nanosystem were used to achieve tumor dynamic photoacoustic imaging and fluorescence imaging after i.v.injection of TSNCs.The tumors bearing mice were irradiated with an 808 nm NIR laser at 24 h after i.v.injection when the uptake level of TSNCs on the tumor site was the highest according to the dynamic imaging results.Under the NIR laser irradiation,the TSNCs could significantly enhance the drug penetration,and kill the cancer cells as a result of chemo-photothermal combination therapy.Part 1:Synthesis,Characterization,and Performance of TSNCs Drug Delivery SystemWe prepared a temperature sensitive multifunctional nanosystem(TSNCs)with an initial size of around 40 nm,which was loaded with NIR agent of copper sulfide nanodots(CuSNDs)and chemotherapeutic drug of doxorubicin(DOX).TSNCs was less toxic than free DOX,which demonstrated that only a limited dose of DOX was released from TSNCs when there was no laser irradiation.TSNCs with laser treatment resulted in significant DNA damage(P<0.001)(y-H2AX staining)and apoptosis rate(P<0.001)(Annexin V/PI staining)than TSNCs treatment due to the release of DOX.The pharmacokinetic in vivo and biological tissue distribution of TSNCs were studied by radioisotope tracer method.The results of pharmacokinetic results showed that the TSNCs had a longer blood circulation ability compared with the small-sized CuSNDs;the biological tissue distribution results showed that TSNCs had a high tunor uptake capacity(8.02%ID/g),which was more than two times higher than CuSNDs(3.62%ID/g).The in vivo photoacoustic imaging and fluorescence imaging revealed that TSNCs had a high accumulation at the tumor site.The multimodality imaging performances also mean that the nanosystem could achieve cancer theranostics.Part 2:Near-infrared Laser Promotes Penetration of TSNCs Drug-loading System in TumorsTo investigate whether the TSNCs + laser result in enhanced DOX penetration in tumor,we used the 3D multicellular spheroids(MCSs)derived from 4T1 breast cancer cells and SKOV3 ovarian cancer as an in vitro model.TSNCs irradiated by laser caused that more DOX was penetrated into the 4T1 and SKOV3 3D tumor spheroids(100 βm from the bottom)than free DOX and TSNCs.The deep tumor penetration effects of TSNCs with laser irradiation were further investigated in 4T1 and SKOV3 solid tumors in vivo.In TSNCs + laser group,the DOX could penetrate to the areas far away from the tumor blood vessels identified by CD34 and the tumor hypoxia areas identified by HIF-la.However,DOX of free DOX and TSNCs groups could only reach around the tumor vessels.Part 3:Study on Anti-tumor Effect of NIR Laser and TSNCs Drug Delivery SystemWe evaluated anticancer efficacy against 4T1 and SKOV3 cancer cells by methyl thiazolyl tetrazolium(MTT)assay in vitro.The experiments included 7 groups:Control,Laser,CuSNDs,TSNCs,DOX,CuSNDs + laser and TSNCs + laser.Obviously,TSNCs with laser treatment showed best anticancer effect between both of 4T1 and SKOV3 cancer cells(P<0.001).Compared with other groups,the treatment of TSNCs + laser led to significantly more apoptosis(P<0.001)(TUNEL staining)and proliferation(P<0.001)(Ki-67 staining)inhibition in the tumors 4T1 and SKOV3 tumor-bearing mice.TSNCs combined with laser inhibited the tumor growth more effectively than other treatments(P<0.05).The in vitro tumorsphere formation assays showed that TSNCs +laser could eradicate breast cancer stem cells(CSCs).Hence,TSNCs with laser treatment inhibited lung and liver metastases of 4T1 cancer cells evaluated by pathological examination.The mice in the DOX group had significantly reduced the left ventricle ejection fraction(EF)and fractional shortening(FS)assessed by echocardiography(P<0.01).TSNCs with laser treatment did not induce EF or FS decline as a result of its tumor targeted drug delivery.Preliminary evaluation of toxic and side effects including blood tests and pathological analysis of major organs indicated the nanosystem was non-toxic after intravenous administration. |
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